Biosensor Analysis of β2-Glycoprotein I–Reactive Autoantibodies: Evidence for Isotype-Specific Binding and Differentiation of Pathogenic from Infection-Induced Antibodies

Abstract

Abstract Background: For the laboratory diagnosis of the antiphospholipid syndrome (APS) we developed a biosensor with the ability to distinguish between disease-relevant anti-β2-glycoprotein I (β2GPI) autoantibodies (anti-β2GPI) and pathogen-specific β2GPI cross-reactive antibodies that occur transiently during infections. Methods: We used a surface plasmon resonance (SPR) biosensor device. For the detection of anti-β2GPI in serum samples, affinity-purified human β2GPI was covalently attached to a functionalized n-alkanethiol self-assembling monolayer on the biosensor chip. After verifying the specificity of the biosensor system with a panel of monoclonal antibodies to β2GPI, we analyzed sera from healthy donors and patients suffering from APS, systemic lupus erythematosus (SLE), syphilis, or parvovirus B19 infections. The SPR results were compared with β2GPI-specific ELISA. Results: Using the SPR biosensor, we recorded antigen binding curves with response levels in the range of 50–500, resonance units (RU) for anti-β2GPI ELISA-positive APS patient sera. The amplitudes of the antiphospholipid antibody (APL) responses in the biosensor correlated with the overall IgG and IgM anti-β2GPI ELISA titers with a correlation coefficient of 0.87. Moreover, we observed immunoglobulin isotype-specific association and dissociation profiles for APL binding of different APS patient sera to the biosensor-immobilized β2GPI. In contrast to APS patient samples, no significant anti-β2GPI binding (response levels <35 RU) was observed in samples from healthy individuals or from patients suffering from SLE, syphilis, or parvovirus B19 infection. Conclusions: The SPR biosensor system enables specific detection of APS-associated β2GPI-reactive APL and differentiation from β2GPI cross-reactive antibodies that occur frequently during acute infections. The established association/dissociation plot for anti-β2GPI responses in APS patient sera gives additional information regarding the influence of anti-β2GPI IgG and IgM isotype distribution.